In cytology, there is an ever-increasing demand for automatic cellular counting, volumetric differentiation and analysis. At the present time a two level screening process manually accomplishes screening of cytological material such as for the detection of cancerous or malignant cells, and for sizing and counting the cells present in a particular amount of material. An observer capable of determining which samples apparently contain abnormal cells and of determining the size cell one hopes to count within a sample first visually prescreens the cells. A trained cytotechnologist or pathologist who makes a final determination as to whether the cells of these samples are inded cancerous then examines the abnormal cell-containing samples. This method fairly accurately finds cancerous cells, but has a number of disadvantages. First, it is slow, requiring considerable technician time. Second, it is costly due to the human time involved. Third, it is nonquantitative in that the criterian of abnormality as well as the amount of cells present in a particular volumetric sample are primarily subjective. Because of the time and costs involved, it is generally not practicable to examine large populations of individuals using these prior art techniques.
It is therefore desirable to have a system for automatically determining the volumetric distribution of a sample of cells, to normalize light signals from a cell analyzer such as that disclosed in U.S. Pat. No. 3,824,402 to Mullaney et al., assigned to the U.S.A. as represented by the U.S.A.E.C.
Presently, prior art electrical analysis devices utilize a simple orifice with two electrodes at either end of the orifice disposed in a surrounding saline solution. An individual cell moving through the orifice displaces some of the conductive fluid in the orifice. Because the conductivity of the cell is less than that of the fluid it displaces, the resistance of the orifice contents increase due to the presence of the cell by an amount related to the volume of the cell. Electrical circuits connected to the electrodes sense this change of resistance and produce a signal pulse. In such a device, the desired signal from the cell mixes with undesired noise signals which originate outside of the orifice, because the sensing electrodes are disposed outside of the orifice, to produce a low signal-to-noise ratio.
Prior art flow systems such as those disclosed in U.S. Pat. No. 3,710,933 to Fulwyler et al., assigned to the U.S.A. as represented by the U.S.A.E.C., U.S. Pat. No. 3,560,754 to Kamentsky, and U.S. Pat. No. 3,675,768 to Sanchez have been applied to these problems in order to provide automatic methods of discriminating and classifying normal and abnormal cells. Flow system analysis, as applied by these prior art systems, allows observation of individual cells as they flow in suspension sequentially through a small detection volume. Large numbers of cells are observed in a short period of time and rapid automatic prescreening procedures are applied. Parameters used in evaluation of the cells are light absorption by the cells, fluorescence emitted by stained cells in response to light incident thereon, cell produced scattering of the light incident on the cells, and the volume of the particles observed.
One of the problems of the prior art systems is that fluorescence detectors currently used with the flow microfluorometers such as disclosed in U.S. Pat. No. 3,824,402 to Mullaney et al., and U.S. Pat. No. 3,710,933 to Fulwyler et al. collect only about 3% of the available fluoresced light.
In accordance with the invention, the ellipsoidal cavity flow cell thereof collects approximately 75% of the available fluorescent light. This provides a signal increase of at least 20 times and an increase in signal-to-noise ratio of at least 10 times over the Mullaney and Fulwyler et al. devices. In practice a signal-to-noise ratio increase of 15 times over the above mentioned systems has been obtained. Thus the system of the invention is highly useful in investigating very weakly fluorescing dyes bound to cells, the fluorescence of very small particles, as well as very small cells and other cells which weakly modulate laser light.